1. Field of the Invention
This invention pertains to the field of serine hydrolases. In particular, this invention pertains to serine hydrolases that have been mutated to introduce one or more cysteines which are then chemically derivatized. These chemically modified mutants demonstrate altered enzymatic activity.
2. Background
Enzymes are now widely accepted as useful catalysts in organic synthesis. However, natural wild-type enzymes do not accept all structures of synthetic chemical interest, nor do they always produce the desired (e.g. enantiomerically pure) products necessary for synthesis. This potential limitation of the synthetic applicabilities of enzymes has been recognized and some progress has been made in altering their specificities in a controlled manner, e.g. using site-directed and random mutagenesis techniques of protein engineering. However, modifying enzyme properties by protein engineering has been generally limited to making natural amino acid replacements. Although molecular biological strategies for overcoming this restriction have recently been derived (Cornish et al. (1995) Angew. Chem. Int. Ed. Engl., 34: 621-633), these procedures are difficult to apply in most laboratories.
In contrast, controlled chemical modification of enzymes offers broad potential for facile and flexible modification of enzyme structure, thereby opening up extensive possibilities for controlled tailoring of enzyme specificity and activity. Changing enzyme properties by chemical modification has been explored previously with early reports by the groups of Bender (e.g. Polgar et al. (1966) J. Am. Chem. Soc., 88: 3153-3154) and Koshland (see, e.g., Neet et al. (1966) Proc. Natl. Acad. Sci., USA, 56: 1606-1611) who created a thiosubtilisin by chemical transformation (CH2OH→CH2SH) of the active site serine residue of subtilisin BPN' to cysteine.
Interest in chemically produced artificial enzymes, including some with synthetic potential was renewed by Wu (see, e.g., Wu et al. (1989) J. Am. Chem. Soc., 111: 4514-4515), Bell et al. (1993) Biochem., 32: 3754-3762), Peterson (see, e.g., Peterson et al. (1995) Biochem., 34: 6616-6620), and more recently Suckling (see, e.g., Suckling et al. (1993) Bioorg. Med. Chem. Lett., 3: 542-534).
U.S. Pat. No. 5,208,158 describes chemically modified detergent enzymes where one or more methionines have been mutated into cysteines. The cysteines are subsequently modified in order to confer upon the enzyme improved stability towards oxidative agents. Although improved stability is often a desirable property, it is also often desirable to alter other enzymatic properties (e.g. specificity, catalytic activity, stereoselectivity, etc.).
Many methods for improving the activity and enantioselectivity of hydrolases have been investigated. They include extreme temperatures (Noritomi et al. (1996) Biotechnol. Bioeng. 51: 95-99; Saka et al. (1997) J. Org. Chem. 62: 4906-4907; Ullmann et al. (1996) Tetrahedron: Asymmetry 7: 2047-2054; Holmberg et al. (1991) Biotechnol. Lett. 13: 323-326; Phillips (1992) Enzyme Microb. Technol. 14: 417-419; Lam et al. (1986) J. Org. Chem. 51: 2047-2050), solvent engineering (Koskinen et al. (1996) Enzymatic Reactions in Organic Media, A. M., Blackie Academic and Professional, London; Gutman et al. (1995) Adv Biochem Eng/Biotechnol 52: 87-128; Griebenow and Klibanov (1997) Biotechnol. Bioeng. 53: 351-362; Bonneau et al. (1993) Bioorg. Chem. 21: 431-438; structural variation of the substrate (Gupta and Kaslauskas (1993) Tetrahedron: Asymmetry 4: 879-888; Sih et al. (1992) Chirality 4: 91-97), imprinting (Rich and Dordick, (1997) J. Am. Chem. Soc. 119: 3245-3252; Russell and Klibanov (1988) J. Biol. Chem. 263: 11624-11626.), lyoprotectants (Dabulis and Klibanov (1993) Biotechnol. Bioeng. 41: 566-571; Khmelnitsky et al. (1994) J. Am. Chem. Soc. 116: 2647-2648), chemical modification (Scouten (1987) Methods Enzymol. 135: 30-78; Polgar and Bender (1966) J. Am. Chem. Soc. 88: 3153-3154; Wu and Hilvert, (1989) Am. Chem. Soc. 111: 4513-4514), site-directed mutagenesis (Wong et al. (1990) J. Am. Chem. Soc., 112: 945-953; Bonneau et al. (1991) J. Am. Chem. Soc., 113: 1026-1030; Zhong et al. (1991) J. Am. Chem. Soc. 113: 683-684; Estell et al. (1985) J. Biol. Chem. 260: 6518-6521; Sears and Wong (1996) Biotechnol. Prog., 12: 423-433), and random mutagenesis (Reetz et al. (1997) Angew. Chem. Int. Ed. Engl. 36: 2830-2832; Chen and Arnold (1993) Proc. Natl. Acad, Sci. USA, 90: 5618-5622; Stemmer (1994) Nature, 370: 389-391). However, the chemical modification of mutant enzymes has been underused as a method for generating new hydrolases with novel properties (Gron et al. (1990) Eur. J. Biochem. 194: 897-901).